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WO2010014975A2 - Utilisation de signatures en micro-arn pour évaluer des niveaux de risque de neuroblastome - Google Patents

Utilisation de signatures en micro-arn pour évaluer des niveaux de risque de neuroblastome Download PDF

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WO2010014975A2
WO2010014975A2 PCT/US2009/052550 US2009052550W WO2010014975A2 WO 2010014975 A2 WO2010014975 A2 WO 2010014975A2 US 2009052550 W US2009052550 W US 2009052550W WO 2010014975 A2 WO2010014975 A2 WO 2010014975A2
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mir
patient
neuroblastoma
risk
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WO2010014975A3 (fr
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Alice L. Yu
You-Chin Lin
Ruey-Jen Lin
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Academia Sinica
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • Neuroblastoma accounting for 15% of pediatric cancer deaths, is a common childhood tumor derived from primitive sympathetic neuroblasts. Based on its plethoric clinical behavior, neuroblastoma can be categorized into two risk groups. High-risk neuroblastoma undergoes malignant tumor progression, while low-risk neuroblastoma either regresses spontaneously or differentiates into benign ganglioneuroma. To achieve high therapeutic efficacy, different treatments shall be applied to patients bearing neuroblastoma tumors with different risk levels.
  • MicroRNAs are a class of small noncoding RNAs that negatively regulate gene expression. These small RNAs are initially produced in cells as long precursors, which are then processed to generate mature miRNAs. Dicer and Drosha are two major endonucleases involved in miRNA processing. miRNAs have been found to play important roles in many physiological processes related to cancer development, e.g., cell proliferation, apoptosis, and differentiation. It has been suggested that miRNAs may serve as prognostic markers and therapeutic targets in cancer treatment.
  • the present invention is based, at least in part, on unexpected discoveries that certain miRNA signatures, optionally in combination with other factors (i.e., Dicer, Drosha, and age at diagnosis), are closely associated with a neuroblastoma patient's risk level or survival/death probability.
  • the present invention features a method of determining the risk level of a neuroblastoma patient based on a 15-biomarker signature, including 12 microRNAs, Dicer, Drosha, and age at diagnosis.
  • This method includes the following steps: (i) obtaining a set of data indicating the expression levels of 12 microRNAs hsa-miRNAs-29a, hsa-miRNAs-30c, hsa-miRNAs-30e, hsa-miRNAs-95, hsa-miRNAs-128a, hsa-miRNAs-128b, hsa-miRNAs-135a, hsa-miRNAs-135b, hsa-miRNAs-137, hsa-miRNAs-138, hsa-miRNAs-148a, and hsa-miRNAs-195 in a neuroblastoma sample of the patient, the expression levels of Dicer and Drosha in the sample, and the patient's age at diagnosis, (ii) processing the set of data by computational analysis to determine a risk pattern, and (iii) assessing the patient's risk level based on the risk pattern.
  • the expression levels of the 12 miRNAs, Dicer, and Drosha can be determined by real-time PCR. In one example, this method is applied to a patient who has not been subjected to clinical staging or any other risk assessment.
  • this invention features a method of assessing the risk level of a neuroblastoma patient based on a 27-miRNA signature.
  • This method includes (i) obtaining a set of data indicating the expression levels of 27 microRNAs, including hsa-miR-149, hsa-miR-129, hsa-miR-27b, hsa-miR-23b, hsa-miR-190, hsa-miR-128a, hsa-miR-15a, hsa-miR-148a, hsa-miR-137, hsa-miR-30c, hsa-miR-197, hsa-miR-195, hsa-miR-26b, hsa-miR-21, hsa-miR-30b, hsa-miR-135a, hsa-miR-126,
  • a signature representing low expression of the 27 miRNAs indicates that the patient is a high-risk neuroblastoma patient and a signature representing high expression of the miRNAs indicates that the patient is a low-risk neuroblastoma patient.
  • the computational analysis is Prediction Analysis of Microarray (PAM) analysis.
  • this invention provides a method of assessing the risk level of a neuroblastoma patient based on a miRNA signature of a neuroblastoma patient.
  • This miRNA signature is determined based on the expression level(s) of one or more of the following miRNAs: hsa-miR-23b, hsa-miR-128a, hsa-miR-15a, hsa-miR-148a, hsa-miR-197, hsa-miR-195, hsa-miR-26b, hsa-miR-21, hsa-miR-135a, hsa-miR-126, hsa-miR-95, hsa-miR-142-5p, hsa-miR-128b, hsa-miR-98, hsa-miR-142-3p, hsa-miR-340,
  • the assessment is made based on a miRNA signature including (a) one or more miRNAs listed above, and (b) one or more of the miRNAs listed below: hsa-miR-149, hsa-miR-129, hsa-miR-27b, hsa-miR-190, hsa-miR-137, hsa-miR-30c, hsa-miR-30b, 5 hsa-miR-30e, hsa-miR-331, and hsa-miR-324-5p.
  • a miRNA signature including (a) one or more miRNAs listed above, and (b) one or more of the miRNAs listed below: hsa-miR-149, hsa-miR-129, hsa-miR-27b, hsa-miR-190, hsa-miR-137, hsa-miR
  • a miRNA signature of a neuroblastoma patient determined by computational analysis, represents low expression of the constituting miRNAs, the patient is assessed as a high-risk patient.
  • the miRNA signature represents high expression of the constituting miRNAs, it indicates that the patient is a low-risk neuroblastoma patient.
  • the present invention also provides a method for predicting a neuroblastoma patient's survival/death probability based on a 20-miRNA signature including hsa-miR-26a, hsa-miR-26b, hsa-miR-27b, hsa-miR-30a-3p, hsa-miR-30e, hsa-miR-95, hsa-miR-128a, hsa-miR-128b, hsa-miR-129, hsa-miR-137, hsa-miR-146, hsa-miR-148a, hsa-miR-149, hsa-miR-152, hsa-miR-186, hsa-miR-190, hsa-miR-197, hsa-miR-324-5p, hsa
  • the invention features a risk assessment method based on the
  • Dicer 2 o expression level profile of Dicer, Drosha, or both, as determined by computational analysis, in a neuroblastoma sample of a patient.
  • a profile representing low expression of Dicer, Drosha, or both indicates that the patient has a high risk level and a profile representing low expression of these two proteins indicates that the patient has a low risk level.
  • the expression level profile of Dicer is determined to assess the risk level of a
  • Also within the scope of this invention is a method of inhibiting neuroblastoma cell growth by administering to a neuroblastoma patient an effective amount of a composition containing (i) a polypeptide including the amino acid sequence of Dicer or Drosha, or a nucleotide sequence encoding the polypeptide, and (ii) a pharmaceutically acceptable carrier.
  • an effective amount refers to the amount of each active agent required to confer therapeutic effect on the patient, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on route of administration, excipient choice, and co-usage with other active agents.
  • the just-described composition can also be used in manufacturing a medicament for inhibiting neuroblastoma cell growth.
  • Fig. 1 shows four risk patterns, Pattern A, Pattern B, Pattern C, and Pattern D, which are determined by PNNSolution analysis based on 15 biomarkers, i.e., 12 miRNAs hsa-miRNAs-29a, hsa-miRNAs-30c, hsa-miRNAs-30e, hsa-miRNAs-95, hsa-miRNAs-128a, hsa-miRNAs-128b, hsa-miRNAs-135a, hsa-miRNAs-135b, hsa-miRNAs-137, 5 hsa-miRNAs-138, hsa-miRNAs-148a, and hsa-miRNAs-195, Dicer, Drosha, and age at diagnosis.
  • A risk patterns A, B, C, and D.
  • B distribution of 66 neuroblastoma patients characteristic for each of the four risk patterns and Kaplan-
  • Fig. 2 includes charts showing the correlations between Dicer/Drosha expression o levels and survival/death probability of neuroblastoma patients.
  • Dicer and probability of event- free survival or overall survival in neuroblastoma patients B: correlation between Drosha and probability of event- free survival or overall survival in neuroblastoma patients.
  • C correlation between Dicer and probability of event-free survival or overall survival in neuroblastoma patients with no MYCN amplification.
  • D correlation 5 between Drosha and probability of event- free survival or overall survival in neuroblastoma patients with no MYCN amplification.
  • Fig. 3 includes tables summarizing correlations between various clinical factors and event-free survival (table A) or overall survival (table B) in neuroblastoma patients via univariate and multivariate Cox regression analysis.
  • Fig. 4 includes tables summarizing correlations between various clinical factors and event- free survival (table A) or overall survival (table B) in neuroblastoma patients with no MYCN amplification via univariate and multivariate Cox regression analysis.
  • Fig. 5 is a diagram showing down-regulation of Dicer and Drosha via RNA interference.
  • A a photo showing reduced expression of Dicer and Drosha, as determined by Westernblot, in neuroblastoma cells Be2C, NMB7, and NB5 transfected with plasmids expressing shRNAs targeting Dicer and Drosha.
  • B a chart showing reduced expression of 5 Dicer and Drosha, determined by quantitative RT-PCR, in the same transfected neuroblastoma cells.
  • C a chart showing down-regulation of miRNA hsa-let7a and hsa-mir-17-5p in the transfected neuroblastoma cells.
  • Fig. 6 is a diagram showing that down-regulation of Dicer and Drosha via RNA interference promotes neuroblastoma cell growth, (a): showing survival rates of o neuroblastoma cells transfected with plasmids expressing shRNAs targeting Dicer and
  • Fig. 7 is a table summarizing survival/death probability prediction based on a 20-miRNA signature including hsa-miR-26a, hsa-miR-26b, hsa-miR-27b, hsa-miR-30a-3p,5 hsa-miR-30e, hsa-miR-95, hsa-miR-128a, hsa-miR-128b, hsa-miR-129, hsa-miR-137, hsa-miR-146, hsa-miR-148a, hsa-miR-149, hsa-miR-152, hsa-miR-186, hsa-miR-190, hsa-miR-197, hsa-miR-324-5p, hsa-miR-331, and hs
  • o Neuroblastoma patients can be categorized into two risk groups, i.e., high-risk and low-risk, based on the behaviors of the tumor they have. See Maris et al., Neuroblastoma Lancet 369(9579):2106-2120, 2007.
  • a neuroblastoma patient's risk level is closely associated with clinical stages and survival/death rates.
  • Tables 1 and 2 below show the different neuroblastoma stages (under the International Neuroblastoma Staging System and 5 the International Neuroblastoma Risk Group Staging System) and their correlations with risk levels:
  • miRNA signatures characterizing the expression level profiles of one or more miRNAs, are reliable markers for assessing a neuroblastoma patient's risk level. More specifically, (a) the 27 miRNAs described in Example 1 below are differentially expressed in high-risk neuroblastoma patients versus low-risk patients and therefore any of the 27 miRNAs or a combination thereof serves as a marker for determining a patient's risk level, (b) the 27 miRNAs as a whole constitute a reliable miRNA signature for assessing the risk level of a neuroblastoma patient (see Example 2 below), and (c) a miRNA signature including the 20 miRNAs described in Example 6 below serves as a reliable marker for predicting a neuroblastoma patient's survival/death probability.
  • the present invention relates to a method to assess a neuroblastoma patient's risk level or survival/death probability based on any of the miRNA signatures mentioned above.
  • a neuroblastoma tumor sample is obtained from a patient (e.g., a Caucasian, an Asian, an African, or a Hispanic) and the expression level(s) of the0 miRNA(s) that constitutes a miRNA signature of interest can be determined by conventional methods.
  • the expression levels are determined by quantitative PCR (also known as real-time PCR) using a kit containing a set of primers specific to the miRNAs to be analyzed.
  • the kit can further contain a pair of primers specific to an internal control RNA, e.g., U6 snRNA.
  • the data indicating miRNA expression levels is first normalized against5 the expression level of the control RNA and the normalized data is then processed by a computational program to generate a miRNA signature (e.g., represented by a numeric number) that characterizes the expression level profile of the miRNAs.
  • This signature is compared with a reference point to determine whether it represents low expression or high expression of the miRNAs.
  • the reference point can be determined based on the miRNA o signatures including the same miRNAs obtained from high-risk and low-risk neuroblastoma patients via computational analysis. For example, it can be the middle point between the signature of high-risk patients and the signature of low-risk patients.
  • the signature When the signature represents low expression of the miRNAs (i.e., similar to that obtained from high-risk neuroblastoma patients), it indicates that the patient has a high risk level.
  • the signature when the signature represents high expression of the miRNAs (i.e., similar to that obtained from low-risk neuroblastoma patients), that patient is determined to have a low risk level.
  • PAM Prediction Analysis of Microarray
  • PNN Plausible Neural Network
  • a 15-Biomarker signature including the 12 miRNAs described in Example 4 below, Dicer, Drosha, and age at diagnosis, is a reliable marker for assessing the risk level of a neuroblastoma patient, in particular, a patient free of clinical 5 staging or any other risk assessment (e.g., MYCN amplification or Shimada histology). Accordingly, the present invention features a risk assessment method using the 15-Biomarker signature as an indicator.
  • the expression levels of the 12 miRNAs, Dicer, and Drosha in the neuroblastoma of a patient can be determined based on the method described above.
  • the data indicating their expression levels and the patient's age at0 diagnosis are processed by a computational program, e.g., PNNSolution, to produce a risk pattern for that patient.
  • This risk pattern can be compared with pre-determined risk patterns representing particular risk levels to determine the patient's risk level. For example, if the risk pattern falls in Pattern A, Pattern C, and Pattern D shown in Fig. 1, it indicates that the patient is at high risk, low risk, and medium-to-low risk, respectively. A patient displaying5 Pattern C or D has a high survival rate. If the risk pattern falls in Pattern B also shown in Fig. 1, it indicates that the patient is likely to be at low-to-medium risk. Further risk assessment would be need to accurately determine that patient's risk level. Assessing Risk Levels Based on Dicer, Drosha, or Both
  • Dicer Dicer
  • Drosha Drosha
  • the expression levels of Dicer and Drosha can be determined following the above-described method and normalized against the expression level of an internal control (e.g., GAPDH or ⁇ -actin).
  • the data thus obtained is processed 5 by a computational program to produce a signature characterizing the expression level of
  • Dicer Dicer, Drosha, or the combination of Dicer and Drosha.
  • This signature is compared with a cut-off value that distinguishes high-risk neuroblastoma patients from low-risk neuroblastoma patients.
  • this cut-off value is obtained by analyzing the expression levels of Dicer and Drosha of high-risk and low-risk patients via student t-test. If o the signature is greater than the cut-off value, representing high expression of Dicer or
  • the patient is determined as having a low risk level; if it is lower than the cut-off value, representing low expression of Dicer or Drosha, the patient is determined as having a high risk.
  • Dicer As the indicator, it can be applied to neuroblastoma patients with no MYCN amplification. Inhibiting Neuroblastoma Cell Growth with Dicer or Drosha
  • Dicer or Drosha can be naturally-occurring proteins from human, swine, mouse, rat, or other species. It also can be a functional variant of any of the naturally-occurring proteins, i.e., having a sequence at least 85% (e.g., 90%, 95%, or 98%) to its wild-type counterpart.
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(17):3389-3402, 1997.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST.
  • any of the above-mentioned polypeptides or nucleic acids can be prepared via conventional methods, e.g., recombinant technology. It can then mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
  • “pharmaceutically acceptable carrier” is a carrier compatible with the activity of the fusion protein (and preferably, stabilizing the activity of the fusion protein) and not deleterious to the subject to be treated.
  • carriers include but are not limited to water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical composition may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.
  • An effective amount of the pharmaceutical composition can be administered to a neuroblastoma patient via a conventional route to suppress neuroblastoma cell growth.
  • Dicer or Drosha polypeptide When a Dicer or Drosha polypeptide is used, it can be dissolved or suspended in the carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or 5 implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • the carrier e.g., physiological saline
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. o Suitable dosages are in the range of 0.01 - 100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compositions available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is5 well understood in the art. Encapsulation of the composition in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
  • a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
  • the just-described pharmaceutical composition can be formulated into dosage forms for different administration routes utilizing conventional methods. For example, it can be o formulated in a capsule, a gel seal, or a tablet for oral administration.
  • Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose.
  • Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the composition with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite.
  • the composition can also be administered in a form of a hard shell 5 tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent.
  • the pharmaceutical composition can be administered via the parenteral route.
  • parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient.
  • Cyclodextrins, or other solubilizing agents well known to those familiar with the art, can be o utilized as pharmaceutical excipients for delivery of the therapeutic agent.
  • the efficacy of the pharmaceutical composition described herein can be evaluated both in vitro and in vivo. Based on the results, an appropriate dosage range and administration route can be determined.
  • EXAMPLE 1 Identification of miRNAs Differentially Expressed in Different Staged Neuroblastoma Tumor
  • the expression levels of 162 miRNAs in the 66 primary neuroblastoma tumors were quantified by real-time PCR using the TaqMan MicroRNA Assays Human Panel-Early Access Kit (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol. Briefly, to amplified each miRNA, 2.5 ng of total RNA (in 15 ⁇ l volume) was subjected to gene-specific reverse transcription, using the TaqMan microRNA Reverse Transcription Kit, followed by q-PCR amplification using miRNA-specific primers, using the 7300 Sequence Detection System (Applied Biosystems).
  • Ct threshold cycle
  • miRNA expression profiles were generated using unsupervised agglomerative hierarchical clustering.
  • Global down-regulation of miRNA expression was observed in advanced neuroblastoma tumors (i.e., INSS stage 4), particularly in advanced tumors with MYCN amplification, as relative to miRNA expression in tumors in the other INSS stages.
  • EXAMPLE 2 Distinguishing High-risk Neuroblastoma Patients from Low-risk Patients 5 Based on a 27-miRNA Signature
  • the expression levels of the 27 miRNAs listed in Table 1 above were found to be associated with a neuroblastoma patient's risk level. More specifically, based on this 27-miRNA signature of the patient as determined by PAM analysis, 23 out of 25 high-risk patients and 26 out of 31 low-risk patients were correctly classified into the o proper risk group, with accuracy of 92% and 84%, respectively. Based on the same miRNA signature, 9 of 10 intermediate -risk samples were classified as low-risk patients. These patients indeed exhibited good clinical outcomes.
  • the expression levels of the 27 miRNAs in the 66 neuroblastoma patients mentioned in Example 1 above were also subjected to PAM analysis. Based on the 27-miRNA 5 signature of these patients, 34 patients were determined as low-risk patient, 5 as intermediate -risk patients, and 22 as high-risk patients. None of the 34 low-risk patients had MYCN amplification in their neuroblastoma and 28 out of the 34 patient were diagnosed at ⁇ 1.5 yr. All of these patients survived. Upon clinical staging, these 34 patients were classified as in INSS stage 1, 2, 3, or 4S. Except for stage 3 patients, those in the other o stages are classified as low-risk patients based on the current Children's Oncology Group
  • EXAMPLE 3 Determining Risk Levels of Neuroblastoma Patients Based on the Expression Levels of Dicer or Drosha
  • Real-time RT-PCR was performed to determine the levels of Dicer and Drosha in 65 of the 66 neuroblastoma tumor samples mentioned in Example 1 above, following the procedures described in Karube et al. Cancer Sci. 96(2): 111-115, 2005). Briefly, 10 ng total RNAs isolated from each of the neuroblastoma samples of the 66 patients were reverse transcribed to cDNAs using SuperscriptTM First-Strand Synthesis System with random 5 hexamer primers (Invitrogen).
  • the cDNAs were then subjected to real-time quantitative PCR in IX SYBR Green Master Mix (Applied Biosystems), using and Dicer-, Drosha- or GAPDH-specif ⁇ c primers as described in Karube et al., Cabcer Sci. 96(2): 111-115, 2005 and Applied Biosystems PRISM 7300-HT. All reactions were performed in triplicate. The expression levels of Dicer and Drosha thus obtained were normalized against the expression o level of GAPDH in the same sample.
  • the expression levels of Dicer and Drosha were then subjected to student t-test to determine a cut-off value that has the highest potential for discriminating two distinct groups, i.e., high-risk group and low-risk group.
  • the results show that the cut-off value for Dicer is about -4.5 and that of Drosha is about -5.13. 5
  • Low expression of Drosha was observed in 82% of neuroblastoma patients in stage 4, in 84% high-risk patients, and in 85% patients bearing MYCN amplification (a high risk indicator).
  • the expression level of Dicer in stage 4 tumors were significantly lower that that in tumors in other stages, particularly in stage 4S (p ⁇ 0.00 ⁇ ).
  • Low expression of Dicer was found to be associated with other high-risk indicators, such as unfavaorable age 0 at diagnosis, later disease stage, MYCN amplification, and Shimada histology (p ⁇ 0.00 ⁇ ,
  • the expression level of Dicer or Drosha was also found to be associated with a patient's survival rate. More specifically, the results obtained from Kaplan-Meier survival analyses show that neuroblastoma patients with low expression of Dicer had a significantly 5 lower event-free survival rate than those with high expression of Dicer (32.4% vs. 79.9%,
  • Dicer was identified as an independent indicator for predicting the overall survival rates/risk levels of neuroblastoma patients with no 5 MYCN amplification.
  • EXAMPLE 4 Assessing Risk Levels of Neuroblastoma Patients Based on a Signature of 150 Biomarkers.
  • the Multivariate Data Clustering and Classification System provided by PNN Technologies, Inc., a unique signature consisting of 15 biomarkers, i.e., miRNAs hsa-miPvNAs-29a, hsa-miRNAs-30c, hsa-miRNAs-30e, hsa-miRNAs-95, hsa-miRNAs-128a, hsa-miRNAs-128b, hsa-miRNAs-135a, hsa-miRNAs-135b, 5 hsa-miRNAs-137, hsa-miRNAs-138, hsa-miRNAs-148a, and hsa-miRNAs-195, Dicer,
  • Drosha Drosha, and age at diagnosis, was identified as an indicator for classifying 65 neuroblastoma patients into four risk groups, each having a risk pattern of Patterns A-D. See Fig. 1.
  • the patients having a risk pattern of Pattern A are all high-risk patients with a death rate of 74%.
  • the patients having a risk pattern of Pattern B can be low, intermediate, or high-risk patients o with the death rate of 12%.
  • Pattern C and Pattern D patients were in either low or intermediate risk levels with 0% death rate.
  • Neuroblastoma cell lines Be2C. NMB7, and NB5 were cultured under the conditions described in Diccianni et al., International Journal of Cancer, 80(l):145-154, 1999 and Lin et o al., Oncogene 26(49):7017-7027, 2007. These cells were transfected with plasmids
  • TRCN0000051262, TRCN0000022253, and TRCN0000072243 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • These plasmids obtained from the National RNAi Core Facility, Genomic Research Center, Academia Sinica, Taiwa, were designed for expressing shRNAs targeting human Dicer, human Drosha, and firefly luciferase (as the negative control).
  • the expression levels of Dicer and Drosha, as well as certain miRNAs, in the transfected cells were determined by routine methods. As shown in Fig. 5, panels A, B, and C, expression of shRNAs targeting Dicer and
  • Drosha successfully reduced Dicer/Drosha expression in transfected neuroblastoma cells.
  • Low expression of Dicer/Drosha also resulted in reduced expression of miRNAs hsa-let7a and hsa-mir-17-5p.
  • cells expressing shRNAs targeting Dicer or Drosha proliferated more rapidly when cultured in a liquid medium and produced more and larger colonies when cultured on a solid medium. See Fig. 6.
  • EXAMPLE 6 Survival-Death Probability Assessment of Neuroblastoma Patients Based on a 20-miRNA Signature
  • PNN Probabililic Neural Network

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Abstract

L'invention concerne des procédés d'évaluation du niveau de risque ou la probabilité de survie/mort d'un patient souffrant de neuroblastome sur la base de plusieurs signatures en micro-ARN, facultativement avec Dicer, Drosha et l'âge au niveau du diagnostic. L'invention concerne également l'utilisation de Dicer, Drosha ou des deux lors de la suppression d'une croissance cellulaire de neuroblastome.
PCT/US2009/052550 2008-08-01 2009-08-03 Utilisation de signatures en micro-arn pour évaluer des niveaux de risque de neuroblastome Ceased WO2010014975A2 (fr)

Priority Applications (1)

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US13/056,043 US20110250192A1 (en) 2008-08-01 2009-08-03 Use of microrna signatures for assessing risk levels of neuroblastoma patients

Applications Claiming Priority (2)

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US13765308P 2008-08-01 2008-08-01
US61/137,653 2008-08-01

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WO2010014975A2 true WO2010014975A2 (fr) 2010-02-04
WO2010014975A3 WO2010014975A3 (fr) 2010-05-06

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US (1) US20110250192A1 (fr)
WO (1) WO2010014975A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103740848A (zh) * 2014-01-28 2014-04-23 厦门大学附属中山医院 基于AllGlo探针荧光定量PCR的hsa-miR-146检测试剂盒及其检测方法
EP3488864A1 (fr) 2013-04-25 2019-05-29 Janssen Vaccines & Prevention B.V. Polypeptides rsv f de pré-fusion solubles stabilisés
WO2019115214A1 (fr) * 2017-12-15 2019-06-20 Universiteit Gent Biomarqueurs pour la charge de maladie du neuroblastome
CN119993262A (zh) * 2025-04-15 2025-05-13 上海晟燃生物科技有限公司 用于神经母细胞瘤自然消退诊断的系统和试剂盒

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015123439A2 (fr) * 2014-02-12 2015-08-20 Duke University Méthodes pour promouvoir la différenciation des neuroblastes et traiter un neuroblastome et pour fournir un pronostic à des sujets atteints d'un neuroblastome
CN110872627A (zh) * 2018-09-04 2020-03-10 复旦大学附属儿科医院 miR-21检测试剂盒及其在判断神经母细胞瘤预后中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2554818A1 (fr) * 2004-02-09 2005-08-25 Thomas Jefferson University Diagnostic et traitement de cancers a l'aide de microarn present dans ou au voisinage de caracteristiques chromosomiennes liees aux cancers
AU2005250432B2 (en) * 2004-05-28 2011-09-15 Asuragen, Inc. Methods and compositions involving microRNA
WO2008125883A1 (fr) * 2007-04-16 2008-10-23 Cancer Research Technology Limited Marqueurs du cancer pour le pronostic et le criblage d'agents anticancéreux

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3488864A1 (fr) 2013-04-25 2019-05-29 Janssen Vaccines & Prevention B.V. Polypeptides rsv f de pré-fusion solubles stabilisés
CN103740848A (zh) * 2014-01-28 2014-04-23 厦门大学附属中山医院 基于AllGlo探针荧光定量PCR的hsa-miR-146检测试剂盒及其检测方法
CN103740848B (zh) * 2014-01-28 2015-10-28 厦门大学附属中山医院 基于AllGlo探针荧光定量PCR的hsa-miR-146检测试剂盒及其检测方法
WO2019115214A1 (fr) * 2017-12-15 2019-06-20 Universiteit Gent Biomarqueurs pour la charge de maladie du neuroblastome
CN119993262A (zh) * 2025-04-15 2025-05-13 上海晟燃生物科技有限公司 用于神经母细胞瘤自然消退诊断的系统和试剂盒
CN119993262B (zh) * 2025-04-15 2025-07-18 上海晟燃生物科技有限公司 用于神经母细胞瘤自然消退诊断的系统和试剂盒

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WO2010014975A3 (fr) 2010-05-06

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